Stomatological disorders in older people: An epidemiological study in the Brazil southern.

BACKGROUND: The aim of this retrospective, cross-sectional and observational study was to perform a survey of the stomatological conditions of elderly patients seen in a period of 40 years at a Stomatology Service in Southern Brazil. MATERIAL AND METHODS: A total of 24,347 medical records were reviewed, of which 5,063 belonged to elderly patients aged 60 to 97 years. The stomatological conditions, systemic conditions, and smoking and alcohol drinking habits as well were recorded. RESULTS: The mean age of the patients was 69.29 years, 67.1% were female and 32.9% were male. Variations of normality accounted for 44.5% of the cases. The most prevalent disorders were fungal infections (26.1%), reactive inflammatory lesions (24.6%), burning mouth syndrome (14.9%), benign neoplasms (12.4%), autoimmune disorders (12.3%), premalignant lesions (10.2%) and malignant epithelial neoplasms (7.2%). Regarding biopsied lesions, squamous cell carcinoma was the most prevalent at 30.2%, followed by hyperplasic lesions (28.2%). CONCLUSIONS: Knowledge of these physiological and pathological conditions in the oral cavity of the older people is essential for early diagnosis and preventive and therapeutic measures when necessary.

Efficient immunization of rhesus macaques with an HCV candidate vaccine by heterologous priming-boosting with novel adenoviral vectors based on different serotypes.

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.

Severe factor X deficiency in pregnancy: case report and review of the literature.

Isolate factor X deficiency is an extremely rare clotting factor disorder inherited in autosomal recessive fashion and pregnancy in a homozygous patient is frequently complicated by recurrent miscarriage, uterine bleeding and premature labour. Eleven pregnancies in seven patients affected by FX deficiency have been reported in the literature. Two additional pregnancies have been reported in a FX variant (FX Friuli). We present a new case of successful at term pregnancy in a homozygous patient.

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation.

Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six- to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay - referred to as repeat-anchored integration capture (RAIC) PCR - that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.

betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles.

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.

Identification and characterization of a novel nuclear factor of activated T-cells-1 isoform expressed in mouse brain.

The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3'-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in T-cells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context.

Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure.

We have investigated the efficacy of a gene transfer strategy based on plasmid DNA electroinjection for the correction of anemia associated with renal failure. An expression plasmid encoding the rat erythropoietin (EPO) cDNA under the control of the CMV promoter as constructed and utilized for this work. Electroinjection of pCMV/rEPO in different rat muscles yielded sustained and long-term EPO production and secretion. The muscle-produced EPO corrected the anemia in five of six nephrectomized rats, used as a model of renal failure. The efficiency of muscle transduction was comparable in rats and mice injected with equivalent amounts of DNA per kilogram of body weight. These results demonstrate that gene electrotransfer can be applied to produce therapeutically significant levels of erythropoietin in chronic renal failure.

Characteristics of the adeno-associated virus preintegration site in human chromosome 19: open chromatin conformation and transcription-competent environment.

Adeno-associated virus (AAV) establishes latency in infected cells by integrating into the cellular genome, with a high preference for a unique region, called AAVS1, of the human chromosome 19. The AAV proteins Rep78 and -68 are postulated to initiate the site-specific integration process by binding to a Rep binding site (RBS) in AAVS1. We provide further evidence to corroborate this model by demonstrating that the AAVS1 RBS in human cell lines is located near a DNase I hypersensitive "open" chromatin region and therefore is potentially easily accessible to Rep proteins. This open conformation is maintained in transgenic rats which carry an AAVS1 3. 5-kb DNA fragment and are proficient for Rep-mediated site-specific integration. Interestingly, the core of the DNAse I hypersensitive site in AAVS1 corresponds to a sequence displaying transcriptional enhancer-like properties, suggesting that AAVS1 constitutes a transcription-competent environment. The implications of our findings for AAV physiology and gene therapy are discussed.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Development of animal models for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.

Impaired Stat3 activation following localized inflammatory stimulus in IL-6-deficient mice.

Interleukin 6 (IL-6) and related gp130-signalling cytokines rapidly activate latent cytoplasmic Stat transcription factors and these are believed to play pivotal roles in the expression of downstream cytokine-responsive genes. We have previously shown in IL-6-deficient (-/-) mice that IL-6 is absolutely required for the transcriptional induction of acute phase response (APR) genes in the liver following localized tissue damage caused by subcutaneous injection of turpentine oil, but is not required when the inflammatory stimulus is administered systemically by intraperitoneal injection of bacterial lipopolysaccharide (LPS). In this paper we show that Stat3 is the only Stat factor induced in liver tissue upon localized inflammatory stimuli, and that its activation is virtually absent in IL-6 deficient mice. During LPS-induced inflammation both Stat1 and Stat3 are activated, and only minor kinetic alterations are detected in IL-6-/- mice. These defects are not due to altered intracellular signal transduction, since they could be complemented by injection of recombinant cytokines. These results establish a direct causal relationship in vivo between Stat activation and acute phase gene expression and define unique functions of IL-6 in Stat3 activation upon localized inflammation.

Interleukin 6 is required for the development of collagen-induced arthritis.

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor alpha (TNF-alpha) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-alpha transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6(-/-) mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.

Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.

There is a growing number of soluble agonistic (IL-6, ciliary neurotropic factor, IL-11, and glia-derived neurotropic factor receptors) and antagonistic (IL-1 and TNF receptors) receptor proteins, modulating the biological functions of their cognate ligands. The physiologic role of these receptor molecules in vivo is unclear. In particular, it is not known how the specificity of function of soluble receptors after release into the blood stream is maintained. We addressed this question by studying the function of the soluble IL-6R (sIL-6R) at the cellular level in the liver. We have generated double transgenic mice coexpressing human sIL-6R and human IL-6 in the liver and have analyzed the expression patterns by in situ hybridization. The expression of the human sIL-6R, driven by the phosphoenolpyruvate carboxykinase promoter, is located mainly in periportal areas, whereas human IL-6 under the control of the metallothionein promoter is uniformly expressed throughout the liver. We show here by in situ hybridization that acute phase protein gene expression induced by human IL-6 and human sIL-6R correlated with the periportal expression of sIL-6R, indicating that sIL-6R acts mainly in an area where it is generated. We conclude that in a concentration-dependent manner, at low concentrations of sIL-6R, there is a predominantly paracrine action at the site of its generation, whereas at higher concentrations of the sIL-6R there are both local and systemic effects.

Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice.

Interleukin 6 (IL-6) is known to inhibit the synthesis of tumour necrosis factor (TNF) in vitro and in vivo. In this study we investigated the possible role of IL-6 as an endogenous inhibitor of TNF production in the brain or in the periphery using IL-6-deficient mice or administering recombinant human IL-6 (rhIL-6). When IL-6-deficient mice were injected intracerebroventricularly (i.c.v.) with lipopolysaccaride (LPS), no differences were observed in the production of TNF in the brain, while in the periphery (serum or spleen) TNF levels were markedly increased (about four-fold). When normal mice were injected i.c.v. with a combination of LPS and rhIL-6, inhibition of TNF production was only slight (about 20%), while IL-6 had a stronger effect (> 80% inhibition) in the periphery. Co-administration of soluble IL-6 receptor (sIL-6R) did not enhance the effect of IL-6 on brain TNF, so this refractoriness cannot be attributed to a lack of IL-6 receptors. Interestingly, IL-6 potently inhibited LPS-induced TNF production by macrophagic cells but not by a microglial cell clone, suggesting that the defective response to IL-6 of the brain lies within the responsiveness TNF producing cells to IL-6. It thus appears that the TNF-inhibitory role of IL-6 is confined to the periphery.

Extramedullary expansion of hematopoietic progenitor cells in interleukin (IL)-6-sIL-6R double transgenic mice.

Soluble cytokine receptors modulate the activity of their cognate ligands. Interleukin (IL)-6 in association with the soluble IL-6 receptor (sIL-6R) can activate cells expressing the gp130 signal transducer lacking the specific IL-6R. To investigate the function of the IL-6-sIL-6R complex in vivo and to discriminate the function of the IL-6-sIL-6R complex from the function of IL-6 alone, we have established a transgenic mouse model. Double-transgenic mice coexpressing IL-6 and sIL-6R were generated and compared with IL-6 and sIL-6R single-transgenic mice. The main phenotype found in IL-6-sIL-6R mice was a dramatic increase of extramedullary hematopoietic progenitor cells in liver and spleen but not in the bone marrow. In IL-6 single-transgenic mice and sIL-6R single-transgenic mice no such effects were observed. The high numbers of hematopoietic progenitor cells were reflected by a strong increase of peripheral blood cell numbers. Therefore, activators of the gp130 signal transducer like the IL-6-IL-6R complex may represent most powerful stimulators for extramedullary hematopoietic progenitor cells. gp130 activators may become important for the expansion of hematopoietic progenitor cells in vivo and in vitro.

Interleukin 6 causes growth impairment in transgenic mice through a decrease in insulin-like growth factor-I. A model for stunted growth in children with chronic inflammation.

Stunted growth is a major complication of chronic inflammation and recurrent infections in children. Systemic juvenile rheumatoid arthritis is a chronic inflammatory disorder characterized by markedly elevated circulating levels of IL-6 and stunted growth. In this study we found that NSE/hIL-6 transgenic mouse lines expressing high levels of circulating IL-6 since early after birth presented a reduced growth rate that led to mice 50-70% the size of nontransgenic littermates. Administration of a monoclonal antibody to the murine IL-6 receptor partially reverted the growth defect. In NSE/hIL-6 transgenic mice, circulating IGF-I levels were significantly lower than those of nontransgenic littermates; on the contrary, the distribution of growth hormone pituitary cells, as well as circulating growth hormone levels, were normal. Treatment of nontransgenic mice of the same strain with IL-6 resulted in a significant decrease in IGF-I levels. Moreover, in patients with systemic juvenile rheumatoid arthritis, circulating IL-6 levels were negatively correlated with IGF-I levels. Our findings suggest that IL-6-mediated decrease in IGF-I production represents a major mechanism by which chronic inflammation affects growth.

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production.

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1 beta and IL-6 itself, we used transgenic mice (NSE-hIL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold), IL-1 beta (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 micrograms LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL-1 were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1 beta or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6, we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.